Alternative Assay Research Articles

Genomes of Potato Mop-Top Virus (Virgaviridae: Pomovirus) Isolates from New Zealand and Their Impact on Diagnostic Methods.

Following the detection of potato mop-top virus (PMTV) in New Zealand in 2018, three near-complete PMTV genomes (AS22, AS99, AS144) were assembled from soil samples taken from potato fields in Canterbury. Phylogenetic analysis revealed that these genomes form a distinct lineage, with limited genetic diversity, within the PMTV species. This analysis supports the hypothesis that these genomes share a common origin, possibly resulting from a single (or limited) incursion of PMTV into New Zealand. A single nucleotide polymorphism was identified in the region where a key diagnostic primer binds. The mismatch of the diagnostic primer has implications for the effectiveness of the Mumford diagnostic protocol currently recommended for use in New Zealand; we recommend that the alternative Pandey assay, for which no primer mismatch was detected, be validated and optimized for use on the viral genomes present in New Zealand.

Looking into the Eyes-In Vitro Models for Ocular Research.

Animal research undoubtedly provides scientists with virtually unlimited data but inflicts pain and suffering on animals. Currently, legislators and scientists alike are promoting alternative in vitro approaches allowing for an accurate evaluation of processes occurring in the body without animal sacrifice. Historically, one of the most infamous animal tests is the Draize test, mainly performed on rabbits. Even though this test was considered the gold standard for around 50 years, the Draize test fails to mimic human response mainly due to human and rabbit eye physiological differences. Therefore, many alternative assays were developed to evaluate ocular toxicity and drug effectiveness accurately. Here we review recent achievements in tissue engineering of in vitro 2D, 2.5D, 3D, organoid and organ-on-chip ocular models, as well as in vivo and ex vivo models in terms of their advantages and limitations.

Circulating tumour DNA - looking beyond the blood.

Over the past decade, various liquid biopsy techniques have emerged as viable alternatives to the analysis of traditional tissue biopsy samples. Such surrogate ‘biopsies’ offer numerous advantages, including the relative ease of obtaining serial samples and overcoming the issues of interpreting one or more small tissue samples that might not reflect the entire tumour burden. To date, the majority of research in the area of liquid biopsies has focused on blood-based biomarkers, predominantly using plasma-derived circulating tumour DNA (ctDNA). However, ctDNA can also be obtained from various non-blood sources and these might offer unique advantages over plasma ctDNA. In this Review, we discuss advances in the analysis of ctDNA from non-blood sources, focusing on urine, cerebrospinal fluid, and pleural or peritoneal fluid, but also consider other sources of ctDNA. We discuss how these alternative sources can have a distinct yet complementary role to that of blood ctDNA analysis and consider various technical aspects of non-blood ctDNA assay development. We also reflect on the settings in which non-blood ctDNA can offer distinct advantages over plasma ctDNA and explore some of the challenges associated with translating these alternative assays from academia into clinical use.

Highly sensitive detection of MUC1 by microchip electrophoresis combining with target recycling amplification and strand displacement amplification

Mucin 1 (MUC1) is usually overexpressed in a variety of malignant tumors, and quantitative analysis of MUC1 plays an important role in the early diagnosis of cancer. In this work, a highly sensitive MUC1 assay was developed by integrating microchip electrophoresis (MCE) with target recycling amplification (TRA) and strand displacement amplification (SDA). Specifically, the presence of MUC1 can trigger the exposure of the designed hairpin probe (HP) to initiate SDA and an amplified amount of ssDNA is produced finally. The amount of these ssDNA can be detected by MCE, then the concentration of MUC1 can be obtained through the correlation between MUC1 concentration and ssDNA concentration. The experimental results show that the MCE signal had a good linear relationship with MUC1 concentration in the range of 1.0 pg/mL - 1.0 × 103 pg/mL with a low limit of detection of 0.23 pg/mL under the optimal conditions (S/N = 3). Additionally, the assay had been successfully applied to detect MUC1 in biological samples with satisfactory results, providing an alternative assay for the detection of other tumor markers owing to the high sensitivity, high selectivity, simple operation and low sample consumption.

An ultra-sensitive electrochemical biosensor using the Spike protein for capturing antibodies against SARS-CoV-2 in point-of-care.

This work presents an innovative ultra-sensitive biosensor having the Spike protein on carbon-based screen-printed electrodes (SPEs), for monitoring in point-of-care antibodies against SARS-CoV-2, a very important tool for epidemiological monitoring of COVID-19 infection and establishing vaccination schemes. In an innovative and simple approach, a highly conductive support is combined with the direct adsorption of Spike protein to enable an extensive antibody capture. The high conductivity was ensured by using carboxylated carbon nanotubes on the carbon electrode, by means of a simple and quick approach, which also increased the surface area. These were then modified with EDC/NHS chemistry to produce an amine layer and undergo Spike protein adsorption, to generate a stable layer capable of capturing the antibodies against SARS-CoV-2 in serum with great sensitivity. Electrochemical impedance spectroscopy was used to evaluate the analytical performance of this biosensor in serum. It displayed a linear response between 1.0 ​pg/mL and 10 ​ng/mL, with a detection limit of ∼0.7 ​pg/mL. The analysis of human positive sera containing antibody in a wide range of concentrations yielded accurate data, correlating well with the reference method. It also offered the unique ability of discriminating antibody concentrations in sera below 2.3 ​μg/mL, the lowest value detected by the commercial method. In addition, a proof-of-concept study was performed by labelling anti-IgG antibodies with quantum dots to explore a new electrochemical readout based on the signal generated upon binding to the anti-S protein antibodies recognised on the surface of the biosensor. Overall, the alternative serologic assay presented is a promising tool for assessing protective immunity to SARS-CoV-2 and a potential guide for revaccination.

Aptamer-Based Biosensors for the Colorimetric Detection of Blood Biomarkers: Paving the Way to Clinical Laboratory Testing.

Clinical diagnostics for human diseases rely largely on enzyme immunoassays for the detection of blood biomarkers. Nevertheless, antibody-based test systems have a number of shortcomings that have stimulated a search for alternative diagnostic assays. Oligonucleotide aptamers are now considered as promising molecular recognizing elements for biosensors (aptasensors) due to their high affinity and specificity of target binding. At the moment, a huge variety of aptasensors have been engineered for the detection of various analytes, especially disease biomarkers. However, despite their great potential and excellent characteristics in model systems, only a few of these aptamer-based assays have been translated into practice as diagnostic kits. Here, we will review the current progress in the engineering of aptamer-based colorimetric assays as the most suitable format for clinical lab diagnostics. In particular, we will focus on aptasensors for the detection of blood biomarkers of cardiovascular, malignant, and neurodegenerative diseases along with common inflammation biomarkers. We will also analyze the main obstacles that have to be overcome before aptamer test systems can become tantamount to ELISA for clinical diagnosis purposes.

Autoantibodies Associated with Autoimmune Liver Diseases in a Healthy Population: Evaluation of a Commercial Immunoblot Test.

Autoantibodies constitute important tools for diagnosing the autoimmune liver diseases (AILD) autoimmune hepatitis and primary biliary cholangitis. The EUROLINE immunoblot assay, detecting multiple specificities, is widely used, but the clinical importance of weakly positive findings is unclear. The manufacturer’s recommended cut-off was evaluated by investigating AILD-associated autoantibodies in 825 blood donors and 60 confirmed AILD cases. Positive findings were followed up with immunofluorescence microscopy on rat tissue, anti-M2-ELISA, alternative immunoblot assay, and liver function tests. Thirty-six (4.4%) blood donors were positive with EUROLINE. The most common specificities were LC-1 (1.6%), gp210 (1.3%), and AMA-M2 (1.1%). In general, the positive results were higher in patients than in blood donors, whereas anti-LC-1 was higher in blood donors. The liver function tests were slightly elevated in 2 of the 36 immunoblot positive blood donors. The majority of the positive EUROLINE findings could not be confirmed with the follow-up tests. The EUROLINE-Autoimmune Liver Diseases-(IgG) immunoblot detected autoantibodies in 4.4% of blood donors without signs of AILD. Our findings indicate that the recommended cut-off can be raised for most specificities without loss of diagnostic sensitivity. The prevalence of anti-LC-1 among blood donors indicates a problem with the antigen source.

An indirect comparison of automated indirect immunofluorescence vs automated solid-phase immunoassays for antinuclear antibody detection: A meta-analysis and adjusted indirect comparison of diagnostic test accuracy.

The primary aim of this study was to conduct a meta-analysis to compare the diagnostic accuracy of automated indirect immunofluorescence (automated-IIF) and fully automated solid-phase immunoassays (solid-phase assays), compared with gold standard conventional manual indirect immunofluorescence (manual-IIF) for antinuclear antibody (ANA) detection. Indirect meta-comparison was performed using prospective studies reporting comparative data between automated-IIF and fully automated solid-phase assays individually to conventional manual-IIF. Diagnostic tests regarding different automated solid-phase assays and automated-IIF for ANA detection were retrieved from the Cochrane Library, PubMed, Embase, Web of Science, Chinese Biological Medicine Database (CBM), China National Knowledge Infrastructure (CNKI), and WANFANG electronic databases from their inception to January 2022. Assessment of the quality of the studies was undertaken using a second version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. The investigated diagnostic indices including pooled sensitivity, pooled specificity, pooled positive likelihood ratio (PLR), pooled negative likelihood ratio (NLR), pooled diagnostic odds ratio (DOR), area under the summary receiver operating characteristic (AUC) of automated-IIF and solid-phase assays, respectively. Relative diagnostic odds ratio (RDOR) was calculated to indirectly compare the diagnostic accuracy of automated-IIF and solid-phase assays. To visualize results, we provide forest plots showing the RDOR with 95% confidence intervals (CI) of the 2 methods against the "gold standard" manual-IIF by R software. Deeks' funnel was used to investigate the publication bias. A total of 16 studies involving 6111 subjects were included in the analysis. The pooled sensitivity, pooled specificity, pooled PLR, pooled NLR, pooled DOR and the AUC were 0.85 (95% CI: 0.84-0.86), 0.82 (95% CI 0.81-0.84), 14.22 (95% CI 8.55-23.65), 0.06 (95% CI 0.03-0.12), 287.0 (95% CI 124.30-662.68) and 0.983 for automated-IIF respectively, and as for solid-phase assays those were 0.73 (95% CI 0.70-0.75), 0.87 (95% CI 0.85-0.89), 5.66 (95% CI 3.33-9.62), 0.30 (95% CI 0.20-0.47), 19.14 (95% CI 8.00-45.79) and 0.894. The results of indirect comparison indicated that automated-IIF had statistically significant higher accuracy for the detection of ANA. This meta-analysis and indirect comparison suggest that automated-IIF should be recommended as an alternative assay for ANA screening under the condition of increased demand for ANA testing in clinical immunology laboratories.

Abstract 2293: Evaluation of multiple myeloma research samples by analysis of B cell heavy and light chain receptors in a single NGS assay

Abstract Introduction: B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. Methods: A B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. Primers are included to amplify rearrangements involving Kappa deletion element. Reproducibility studies and clonality assessment were conducted using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal via flow cytometry or IHC/ISH in tissue sections. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results: Clonality assessment of MM research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples contained somatic hypermutation with 6 of 45 samples showing mutation rates greater than 10%. Lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Conclusions: These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify workflow and inclusion of analysis tools such as automated somatic hypermutation rate determination and clonal lineage identification to open new paths for research in B cell disorders. Citation Format: Geoffrey Marc Lowman, Landon Pastushok, Karen Mochoruk, Wayne Hill, Michelle Toro, Loni Pickle, Carolina Gonzalez, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang, Julie Stakiw, Mark Bosch, Hadi Goubran, Ron Geyer, John DeCoteau. Evaluation of multiple myeloma research samples by analysis of B cell heavy and light chain receptors in a single NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2293.

A High-Throughput Yellow Fever Neutralization Assay.

ABSTRACTQuick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics.IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

Plasma MIA, CRP, and Albumin Predict Cognitive Decline in Parkinson's Disease.

Using a multi-cohort, discovery-replication-validation design, we sought new plasma biomarkers that predict which individuals with Parkinson's disease (PD) will experience cognitive decline. In 108 discovery cohort PD individuals and 83 replication cohort PD individuals, we measured 940 plasma proteins on an aptamer-based platform. Using proteins associated with subsequent cognitive decline in both cohorts, we trained a logistic regression model to predict which patients with PD showed fast (>=1 point drop/year on Montreal Cognitive Assessment [MoCA]) versus slow (< 1 point drop/year on MoCA) cognitive decline in the discovery cohort, testing it in the replication cohort. We developed alternate assays for the top 3 proteins and confirmed their ability to predict cognitive decline - defined by change in MoCA or development of incident mild cognitive impairment (MCI) or dementia - in a validation cohort of 118 individuals with PD. We investigated the top plasma biomarker for causal influence by Mendelian randomization (MR). A model with only 3 proteins (melanoma inhibitory activity protein [MIA], C-reactive protein [CRP], and albumin) separated fast versus slow cognitive decline subgroups with an area under the curve (AUC) of 0.80 in the validation cohort. The individuals with PD in the validation cohort in the top quartile of risk for cognitive decline based on this model were 4.4 times more likely to develop incident MCI or dementia than those in the lowest quartile. Genotypes at MIA single nucleotide polymorphism (SNP) rs2233154 associated with MIA levels and cognitive decline, providing evidence for MIA's causal influence. An easily obtained plasma-based predictor identifies individuals with PD at risk for cognitive decline. MIA may participate causally in development of cognitive decline. ANN NEUROL 2022;92:255-269.

Alternative Assay Reagents for UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module

This report advises against the use of 5-iodoridine or 5-ethynyluridine as alternative assay reagents in the PUB module, primarily due to their lack of an isosbestic point of phosphorolysis under moderately alkaline conditions.

Development of functionally relevant potency assays for monovalent and multivalent vaccines delivered by evolving technologies.

A potency or potency-indicating assay is a regulatory requirement for the release of every lot of a vaccine. Potency is a critical quality attribute that is also monitored as a stability indicator of a vaccine product. In essence, a potency measurement is a test of the functional integrity of the antigen and is intended to ensure that the antigen retains immunocompetence, i.e., the ability to stimulate the desired immune response, in its final formulation. Despite its central importance, there is incomplete clarity about the definition and expectation of a potency assay. This article provides a perspective on the purpose, value, and challenges associated with potency testing for vaccines produced by new technologies. The focus is on messenger RNA vaccines in the light of experience gained with recombinant protein-based vaccines, which offer the opportunity to directly correlate in vitro antigenicity with in vivo immunogenicity. The challenges with developing immunologically relevant in vitro assays are discussed especially for multivalent vaccine products, the importance of which has been reinforced by the ongoing emergence of SARS-CoV-2 variants of concern. Immunoassay-based release of multivalent vaccine products, such as those containing multiple antigens from different variants or serotypes of the same virus, require antibodies that are selective for each antigen and do not significantly cross-react with the others. In the absence of such exclusively specific antibodies, alternative functional assays with demonstrable correlation to immunogenicity may be acceptable. Initiatives for geographically distributed vaccine technology facilities should include establishing these assay capabilities to enable rapid delivery of vaccines globally.

Screening and Identification of HBV Epitopes Restricted by Multiple Prevalent HLA-A Allotypes.

Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.

Dubiously increased FT4 and FT3 levels in clinically euthyroid patients: clinical finding or analytical pitfall?

We systematically investigated normally or subclinically increased thyroid stimulating hormone (TSH) values associated with unexpectedly increased thyroxine (FT4) and free triiodothyronine (FT3) in findings of patients without any thyroid disease. Moreover, we looked for alternatives to overcome such states with an improved diagnostic procedure and to investigate the pathogenetic background of the respective patients. Samples with TSH concentrations within the range of 0.4-10mU/L combined with increased concentrations of FT4 (n=120; Cobas, Roche) were collected over a period of around six years. Cobas FT4 results were compared with measurements from Liaison (DiaSorin) and Architect (Abbott) FT4 assays. For further validation all samples were measured for total thyroxine (TT4) (Cobas, Roche). Finally, FT3 and TT3 as complementary parameters were measured in samples with leftover material. To overcome potential analytical disturbances from stimulating heterophilic antibodies, we used heterophilic blocking tubes (HBTs). From the 120 samples with increased FT4 concentrations by Cobas, 51/120 were also increased by Liaison, and 26/120 by Architect. However, the measurement of TT4 indicated only n=10/120 increased values. The number of increased FT3 (n=71) measurements was higher in Architect>Cobas>Liaison (28>27>9). TT3 levels of 70/71 samples were within the reference interval. HBTs were inappropriate to reduce unspecific immunoreactivity in our samples. No clear pathogenetic background could be elucidated in the anamnesis of individual patients. To overcome dubious constellations of TSH, FT4, and FT3, it is helpful to measure TT4 and TT3 for control or to use an immunoassay with an alternative assay design for the respective parameters.

Weight of evidence tools in the prediction of acute fish toxicity.

Acute fish toxicity (AFT) is a key endpoint in nearly all regulatory implementations of environmental hazard assessments of chemicals globally. Although it is an early tier assay, the AFT assay is complex and uses many juvenile fish each year for the registration and assessment of chemicals. Thus, it is imperative to seek animal alternative approaches to replace or reduce animal use for environmental hazard assessments. A Bayesian Network (BN) model has been developed that brings together a suite of lines of evidence (LoEs) to produce a probabilistic estimate of AFT without the testing of additional juvenile fish. Lines of evidence include chemical descriptors, mode of action (MoA) assignment, knowledge of algal and daphnid acute toxicity, and animal alternative assays such as fish embryo tests and in vitro fish assays (e.g., gill cytotoxicity). The effort also includes retrieval, assessment, and curation of quality acute fish toxicity data because these act as the baseline of comparison with model outputs. An ideal outcome of this effort would be to have global applicability, acceptance and uptake, relevance to predominant fish species used in chemical assessments, be expandable to allow incorporation of future knowledge, and data to be publicly available. The BN model can be conceived as having incorporated principles of tiered assessment and whose outcomes will be directed by the available evidence in combination with prior information. We demonstrate that, as additional evidence is included in the prediction of a given chemical's ecotoxicity profile, both the accuracy and the precision of the predicted AFT can increase. Ultimately an improved environmental hazard assessment will be achieved.Integr Environ Assess Manag 2023;19:1220-1234. © 2022 SETAC.

Looking at Developmental Neurotoxicity Testing from the Perspective of an Invertebrate Embryo.

Developmental neurotoxicity (DNT) of chemical compounds disrupts the formation of a normal brain. There is impressive progress in the development of alternative testing methods for DNT potential in chemicals, some of which also incorporate invertebrate animals. This review briefly touches upon studies on the genetically tractable model organisms of Caenorhabditis elegans and Drosophila melanogaster about the action of specific developmental neurotoxicants. The formation of a functional nervous system requires precisely timed axonal pathfinding to the correct cellular targets. To address this complex key event, our lab developed an alternative assay using a serum-free culture of intact locust embryos. The first neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons which use guidance cues from membrane-bound and diffusible semaphorin proteins. In a systematic approach according to recommendations for alternative testing, the embryo assay quantifies defects in pioneer navigation after exposure to a panel of recognized test compounds for DNT. The outcome indicates a high predictability for test-compound classification. Since the pyramidal neurons of the mammalian cortex also use a semaphorin gradient for neurite guidance, the assay is based on evolutionary conserved cellular mechanisms, supporting its relevance for cortical development.

HPV E1 qPCR, a Low-Cost Alternative Assay to Roche Diagnostic Linear Array is Effective in Identifying Women at Risk for Developing Cervical Cancer.

ObjectiveSince a quantitative polymerase chain reaction (qPCR) assay targeting the E1 region of HPV genome is cost-effective/simple to perform, we evaluated the agreement between the Roche Diagnostics Linear Array (RDLA) genotyping test and qPCR-based E1 assay to detect HR-HPV genotypes that are included or not included in HPV vaccines and compared their accuracy to detect CIN 2+.MethodsStudy population included 257 African American (AA) and 266 Caucasian American (CA) diagnosed with intraepithelial neoplasia (CIN) grades ≤CIN 1 or ≥CIN 2 (CIN 2+) and tested for HPV by the RDLA and E1 assay. The concordance was determined using Gwet’s AC1. The calculated positive predictive value (PPV) and negative predictive value (NPV) of the two assays were used to determine their suitability to detect CIN lesions.ResultsOverall, the E1 assay showed substantial agreement with the RDLA assay to detect any HR-HPV genotype and the agreement was higher in women diagnosed with CIN 2+ than ≤CIN 1. The concordance was largely higher in Cas than in Aas. The NPV and PPV values to detect CIN lesions were similar between the two assays.ConclusionUtilization of the HPV E1 assay as a tool for CC screening could be a cost-effective approach that applies to both vaccinated and unvaccinated populations.

A sensitive and less cytotoxic assay for identification of proliferating T cells based on bioorthogonally-functionalized uridine analogue

Quantitative detection of T cell proliferation is an important readout in immunology research, as it is one of the hallmarks of T cell activation. Fluorescence-based methods for T cell proliferation mostly rely on the usage of probes that non-specifically conjugate to free primary amine groups in cells. Each cell division then results in a two-fold dilution of the probes which is detectable with flow cytometry. However, questions have been raised about cytotoxicity of these dilution-based T cell proliferation probes and they potentially affect T cell activation. An alternative assay relies on the incorporation of the uridine analog BrdU in the DNA of dividing T cells that can be detected with an antibody, but this requires harsh fixation and denaturation conditions. Recently, a new assay for detection of cell proliferation has been developed, based on the incorporation of the bioorthogonally-functionalized uridine analog 5-ethynyl-2′-deoxyuridine (EdU). Goal of this study was to compare the sensitivity and cytotoxicity of the EdU assay with a widely-used dilution-based T cell proliferation probe, CellTrace Far Red. We found that, compared to the dilution-based probe, the EdU-based assay better preserves T cell viability, is more sensitive for detecting T cell proliferation, and allows for better discernable interferon gamma responses.

Within- and between-laboratory reproducibility and predictive capacity of amino acid derivative reactivity assay (ADRA) using a 0.5 mg/mL test chemical solution: Results of the study for reproducibility confirmation implemented in five participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.